Pharmaceutical formulations for the treatment of pulmonary arterial hypertension

ABSTRACT

Pharmaceutical formulations are described for use in the treatment of pulmonary arterial hypertension (PAH). The formulations comprise polymeric nanoparticles encapsulated within crosslinked polymeric hydrogel microparticles, wherein the polymeric nanoparticles carry a therapeutic agent suitable for treatment of PAH loaded within them (for example, prostacyclin synthetic analogs, PPAR β agonists and NO donors). Preferred formulations are inhalable, dry powder pharmaceutical formulations, which are able to swell on administration to the lungs of a patient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on and claims priority to United KingdomApplication No. 1400412.1, filed on Jan. 10, 2014, the entire contentsof which is fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical formulations for thetreatment of pulmonary arterial hypertension (PAH). Aspects of theinvention relate to inhalable, dry powder formulations for pulmonaryadministration. Further aspects of the invention relate to formulationssuitable for injectable or oral administration.

BACKGROUND TO THE INVENTION

Pulmonary arterial hypertension (PAH) is a rare but devastating disease,in which the normally low pulmonary artery pressure becomes elevated dueto vaso-constriction and to the remodelling of pulmonary vessels. Thisin turn increases workload on the right side of the heart, causing rightheart hypertrophy, fibrosis and ultimately heart failure.

Interventions used in the management of PAH are traditionally targetedon the vasculature, with the aim of enhancing vasodilation andanti-proliferation pathways. These include the prostacyclin analoguesand nitric oxide (NO). However, it is increasingly recognized that inaddition to the pulmonary vasculature, the right heart is also a viabletherapeutic target in the treatment of PAH. PPAR β inhibitors have beenshown in recent studies on animal models to reduce right hearthypertrophy without influencing pulmonary vascular remodelling.

Prostacyclin Analogues

Prostacyclin is a powerful vasodilator, produced in the body byendothelial cells. Patients with PAH are found to have low levels ofprostacyclin, leading to a frequently life-threatening constriction ofthe pulmonary vasculature.

Natural prostacyclin has been found to be unstable in solution, andundergoes rapid degradation, making it very difficult to use forclinical applications. Over 1,000 synthetic prostacyclin analogues havebeen developed to date as a result. One of these, Treprostinil hasdemonstrated unique effectiveness in inhibiting platelet activation andas a vasodilator, and it has relatively good stability in solutioncompared to native prostacyclin. Other prostacyclin analogues, such asSelexipag are currently undergoing clinical trials with highlyencouraging results.

Treprostinil is currently marketed in two formulations, as an infusion(subcutaneous or intravenous via a continuous infusion pump), or as aninhaled aerosol, used with a proprietary device, 4 times per day and atleast 4 hours apart. The infusion is frequently associated with sideeffects such as severe site pain or reaction, whilst the aerosolrequires a disciplined regimen on the part of patients. Attempts todevelop an oral formulation have to date failed FDA regulatory approvaldue to the relatively adverse risk/efficiency ratio of prototypes triedso far.

Developing alternative formulations of Treprostinil, which bypass themany serious shortcomings of the continuous infusion/pump system, andwhich allow efficient, controlled, targeted and sustained release of thedrug without the complexity of the current aerosol system, shouldimprove patient compliance and experience of treatment, and ultimatelyimpact on successful management of the condition.

Selexipag is currently undergoing Phase III trials. Selexipag isdesigned to act on IP (prostacyclin) receptors selectively, and has todate demonstrated a significant reduction in pulmonary vascularresistance within the trial cohorts studied. It is currently produced asan oral formulation, and although the administration regime is farsimpler than for Treprostinil, again the development of a formulationand delivery mechanism that allows efficient, controlled, targeted andsustained release of the drug would significantly reduce side effectsand improve patient compliance, ultimately impacting positively onoutcome.

Nitric Oxide

Nitric oxide is known to have vasodilatory effects on the pulmonaryvasculature in both humans and animals. A number of trials suggest thatthe effects of NO on vascular resistance are selective (ie focused onpulmonary rather than systemic), and do not cause systemic hypotensionor raise methaemoglobin.

The main challenge with NO therapy is in sustaining benefit over aprolonged period. A delivery mechanism that allows controlled, sustainedrelease of NO, such as that offered by the present invention, cansubstantively overcome this challenge. Formulations of NO donors (suchas, but not limited to, organic nitrates, nitrite salts,s-nitrosoglutathione (GSNO), and S-Nitrosothiols) can be loaded into thedelivery mechanism, which can produce sustained and controlled doses ofNO when activated by the moisture in the lung.

PPAR β Agonists

The pulmonary vasoconstriction and remodelling associated with PAH leadto overloading and hypertrophy of the right heart, and eventually toheart failure. While focusing on vasodilatation is an important part ofeffective management of PAH, it is increasingly apparent that thehypertrophic right heart is also a valid therapeutic target forintervention in the disease.

The PPARs (peroxisome proliferator activated receptors) have been shownin studies to be an attractive protective pathway in the overloadedheart. As well as protecting against ischemia reperfusion injury, inmice, PPAR has demonstrated effects on reducing left ventriculardilation, fibrosis and mitochondrial abnormalities. GW0742, a ligandwhich activates PPAR β selectively, has been shown in studies to reduceright heart hypertrophy without influencing pulmonary vascularremodelling. GW0742 is not currently used in the treatment of PAH.Nonetheless it is a feature of this invention that suitable formulationsof GW0742 can be developed for loading into nano-particle deliverysystems for lung, oral and intravascular administration.

Current treatments for PHA require that the therapy is administered byinjection, or by inhalation. Inhalation would typically be preferredover injection, but current inhalable therapies require complexadministration forms and schedules, and this has an effect on patientcompliance or ease of delivery. Some therapeutics (for example, NOdonors) require sustained release for effective administration.

The present invention proposes alternative formulations, which in someembodiments would be easier to administer.

BRIEF SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is providedan inhalable, dry powder pharmaceutical formulation for the treatment ofpulmonary arterial hypertension (PAH), the formulation comprisingpolymeric nanoparticles encapsulated within crosslinked polymericmicroparticles, wherein the polymeric nanoparticles carry a therapeuticagent suitable for treatment of PAH loaded within them.

By “nanoparticle” is meant a composition having a mean particle size(preferably diameter) of less than 600 nm, preferably less than 500 nm.Preferably the mean diameter ranges from 1 to 500 nm, more preferably10-250 nm. In preferred embodiments, the mean diameter is less than 250nm, preferably less than 200 nm.

By “microparticle” is meant a composition having a mean particle size(preferably diameter) ranging from 0.75 to 10 μm. Preferred meanparticle size ranges include 0.75 to 7.5 μm, 1 to 5 μm, 2 to 4 μm. Mostpreferred range is 1 to 5 μm.

The mean particle size may be determined by any suitable methodpracticed in the art; examples of suitable methods are exemplifiedherein.

The nanoparticle plus microparticle formulation described herein may bereferred to as a nano-micro-carrier, or nano-micro-particles, ornano-micro formulation.

The present formulations provide therapeutic routes for treatment of PAHwhich are simpler to administer than existing treatments. This isachieved by incorporating the drug into nanoparticles. However,nanoparticles themselves are difficult to administer by inhalation, sothese are then incorporated into crosslinked polymeric microparticles.This makes the formulation suitable for dry powder inhalation. Thenanoparticles encapsulate and stabilise the drug, while also allowingfor pulmonary delivery due to their size. The microparticles can help tomake the physical form more suitable for one or another administrationroute, while also permitting sustained release of the nanoparticles.

The nanoparticles and microparticles are preferably biodegradable, andare preferably biocompatible. By biodegradable is meant that theparticles will break down naturally within the body under physiologicalconditions; preferably the conditions as found within the lung. Bybiocompatible is meant that the particles will not elicit an immuneresponse from the patient. The nanoparticles preferably biodegrade underphysiological conditions to give a drug release rate of 0.1-0.3 w % perhour.

The polymeric nanoparticles preferably comprise a chitosan or achitosan-derivative polymer. Suitable chitosan-derivative polymersinclude chitosan-PEG, N-trimethyl chitosan, and/or chitosan derivativeshaving hydrophobic side chains (for example, stearic acid, cholanicacid, phthaloyl, butyl acrylate). Alternatively, the nanoparticles maycomprise a copolymer, preferably an amphiphilic copolymer, for example,poly(lactic-co-glycolic acid) (PLGA). In preferred embodiments, thenanoparticles are graft and block copolymers. Chitosan or chitosanderivatives are particularly preferred, due to chitosan's ability toenhance absorption in lung tissues through opening the intercellulartight junctions of the lung epithelium. We refer to this ability hereinas “epithelial targeting”, and this feature also makes similarformulations suitable for administration to other epithelial tissues,for example, the intestine. Thus, in preferred embodiments, thenanoparticles are epithelially targeted.

The polymeric nanoparticles in preferred embodiments have a moisturecontent, in the dry formulation, of less than 2%.

The polymeric nanoparticles may be produced via emulsion polymerization,ionotropic gelation, polyelectrolyte complexation, and/or self-assembly.In preferred embodiments, the nanoparticles may be produced via selfassembly following sonication of amphiphilic polymer solutions.

The cross-linked polymeric microparticles preferably comprisecross-linked hydrogel polymers, and are preferably crosslinked hydrogelmicroparticles. These may be in the form of semi-interpenetratingpolymeric networks (semi-IPNs) and full-IPNs. These semi- and full-IPNsare preferably based on natural polymers such as, but not limited to,chitosan and water soluble chitosan derivatives (such as carboxymethyland PEGylated derivatives) in a combination with one or more ofnontoxic, biocompatible, and biodegradable polymers including, but notlimited to, hyaluronate, carrageenan and oligoguluronate. In someembodiments, only chitosan or chitosan derivatives are used. Thesemi-IPN and IPN microparticles are crosslinked through any suitablemethod, including ionotropic gelation, polyelectrolyte complexationand/or H-bonding. The nanoparticles-microparticles formulations may beproduced using a spray-drying technique, spray gelation, or ionotropicgelation followed by lyophilization. Spray drying is preferred.

The microparticles are preferably swellable; more preferably themicroparticles are hydrogel microparticles and the hydrogel isswellable. This allows the hydrogel to absorb moisture from the lung orother delivery site and so permit release of the nanoparticles. Thehydrogel is preferably able to swell to at least 200, 300, 400, 500% ofthe original (dry formulation) size. In a preferred embodiment, amicroparticle of 2-5 μm dry diameter is able to swell to at least 20 μmdiameter. The microparticle is preferably able to swell to the largerdiameter within 10, 9, 8, 7, 6, 5, 4, 3, or 2 minutes fromadministration to the lungs of a patient. In a further preferredembodiment, the microparticle is able to swell to at least 10 times thedry size within 3 minutes from administration.

The hydrogel preferably comprises less than 10%, preferably less than7.5%, more preferably less than 5, 4, 3, 2% water when in the dryformulation. In preferred embodiments, this is less than 2%.

The particles preferably biodegrade under physiological conditions (3-5%weight loss per day) to give a drug release rate of less than 1% perhour, more preferably less than 0.5% per hour. In preferred embodiments,the drug release rate is 0.1-0.3 w % per hour.

The microparticles may comprise a pH responsive carrier. SmartpH-responsive particles for drug delivery are known, and are used insituations where it is desirable to release a drug from a carrier undercertain pH conditions; for example, when the carrier is in a specificenvironment, such as the intestine. Examples of the preparation and useof pH-responsive carriers are given in PS Stayton and AS Hoffman, “SmartpH-responsive carriers for intracellular delivery of biomoleculardrugs”, in V Torchilin (ed), Multifunctional PharmaceuticalNanocarriers, Springer Science and Business Media, 2008; and inStephanie J. Grainger and Mohamed E. H. El-Sayed, “STIMULI-SENSITIVEPARTICLES FOR DRUG DELIVERY”, in Biologically Responsive HybridBiomaterials, Esmaiel Jabbari and Ali Khademhosseini (Ed), Artech House,Boston, Mass., USA.

The therapeutic agent is preferably selected from prostacyclin syntheticanalogs, PPAR β agonists and NO donors. The prostacyclin syntheticanalog may be treprostinil or selexipag. The PPAR β agonist may beGW0742. In preferred embodiments, the agent is a prostacyclin syntheticanalog, most preferably treprostinil.

The dosage of the therapeutic agent may be selected depending on thedesired administration dose to the patient, and may vary depending onthe agent to be used, and the composition of the nano andmicroparticles.

According to a further aspect of the invention, there is provided acomposition comprising polymeric nanoparticles encapsulated withincrosslinked polymeric microparticles, wherein the polymericnanoparticles carry a therapeutic agent suitable for treatment of PAHloaded within them, for use in the treatment of pulmonary arterialhypertension (PAH). The invention also provides the use of a compositioncomprising polymeric nanoparticles encapsulated within crosslinkedpolymeric microparticles, wherein the polymeric nanoparticles carry atherapeutic agent suitable for treatment of PAH loaded within them inthe manufacture of a medicament for the treatment of PAH.

A further aspect of the invention provides a method of treatment of PAH,the method comprising administering a composition comprising polymericnanoparticles encapsulated within crosslinked polymeric microparticles,wherein the polymeric nanoparticles carry a therapeutic agent suitablefor treatment of PAH loaded within them to a patient in need thereof,wherein the composition is administered by inhalation.

A further aspect of the invention provides a pharmaceutical formulationcomprising polymeric nanoparticles encapsulated within smartpH-responsive crosslinked polymeric microparticles, wherein thepolymeric nanoparticles carry a therapeutic agent selected fromprostacyclin synthetic analogs, PPAR β agonists and NO donors loadedwithin them, wherein the pH-responsive microparticles are targeted tothe intestine, and wherein the nanoparticles are targeted to theepithelium. This aspect of the invention may be suitable for intestinalor oral administration, for example. Smart pH-responsive particles fordrug delivery are known, and are used in situations where it isdesirable to release a drug from a carrier under certain pH conditions;for example, when the carrier is in a specific environment, such as theintestine. Examples of the preparation and use of pH-responsive carriersare given in PS Stayton and AS Hoffman, “Smart pH-responsive carriersfor intracellular delivery of biomolecular drugs”, in V Torchilin (ed),Multifunctional Pharmaceutical Nanocarriers, Springer Science andBusiness Media, 2008; and in Stephanie J. Grainger and Mohamed E. H.El-Sayed, “STIMULI-SENSITIVE PARTICLES FOR DRUG DELIVERY”, inBiologically Responsive Hybrid Biomaterials, Esmaiel Jabbari and AliKhademhosseini (Ed), Artech House, Boston, Mass., USA.

A yet further aspect of the invention provides an injectablepharmaceutical formulation comprising polymeric nanoparticles carrying atherapeutic agent selected from prostacyclin synthetic analogs, PPAR βagonists and NO donors loaded within them, and wherein the nanoparticlesare targeted to the epithelium. For injectable formulations, it may notbe beneficial to provide microparticles, and the nanoparticles may beinjected directly. Although not preferred for treatment of PAH, suchformulations may find application where inhalation is not practical forone reason or another, or where sites other than the lungs are to betargeted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows synthesis of PEG-CS copolymers

FIG. 2 shows synthesis of PEG-CS-oleic and PEG-CS-cholanic copolymers

FIG. 3 shows characterization and NO release profile from NOnanoparticles

FIG. 4 shows the effect of NO nanoparticles on viability and chemokinerelease from endothelial cells

FIG. 5 shows the effect of NO nanoparticles on viability and chemokinerelease from arterial smooth muscle cells

FIG. 6 shows the effect of NO nanoparticles on relaxation of pulmonaryarteries in mice

FIG. 7 shows the effect of control nanoparticles on viability andchemokine release from endothelial cells

FIG. 8 shows the effect of U46619 on pulmonary arteries in mice, and theeffect of control nanoparticles on mice

FIG. 9 shows the effect of U46619 on pulmonary arteries in mice, and theeffect of control nanoparticles and NO nanoparticles on mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel formulations of therapeutics forthe treatment of PAH. Preferred therapeutic agents include prostacyclinsynthetic analogs, PPAR β agonists and NO donors. The agents areincorporated into biodegradable polymeric nanoparticles, whichthemselves are incorporated into hydrogel microparticles or smartpH-responsive microparticles.

Background art which may be of benefit in understanding the inventionincludes:

-   El-Sherbiny, I. M., & Smyth, H. D. C. (2012). Controlled release    pulmonary administration of curcumin using swellable biocompatible    nano-microparticles systems. Molecular Pharmaceutics, 9(2), 269-280.-   El-Sherbiny I M, and Smyth, H D C. (2010) Biodegradable nano-micro    carrier systems for sustained pulmonary drug delivery: (I)    self-assembled nanoparticles encapsulated in respirable/swellable    semi-IPN microspheres. Int J. Pharm 395: 132-141.-   El-Sherbiny I M, Mcgill S, and Smyth H D C. (2010) Swellable    microparticles as carriers for sustained pulmonary drug delivery. J.    Pharm Sci, 99(5): 2343-2356.-   El-Sherbiny I M, and Smyth H D C. (2010) Novel cryomilled physically    crosslinked biodegradable hydrogel microparticles as carriers for    inhalation therapy. J. Microencapsulation, 27(7): 561-572.-   El-Sherbiny I M, Abdel-Mogibb M, Dawidar A, Elsayed A, and Smyth H    D C. (2010) Biodegradable pH-responsive    alginate-poly(lactic-co-glycolic acid) nano/micro hydrogel matrices    for oral delivery of silymarin, CarbohydrPolym, 83, 1345-1354.-   El-Sherbiny I M, and Smyth H D C. (2010) PLGA nanoparticles    encapsulated in respirable/swellable hydrogel microspheres as    potential carriers for sustained drug delivery to the lung. Annual    Meeting of American Association of Pharmaceutical Scientists, New    Orleans, La.-   El-Sherbiny I M, and Smyth H D C. (2010) Nano-micro carrier systems    for sustained pulmonary drug delivery. Biomedical Engineering    Society Annual Meeting (BMES), Austin, Tex.-   Selvam P, El-Sherbiny I M, and Smyth H D C. (2010) Swellable    microparticles for sustained release drug delivery to the lung using    propellant driven metered dose inhalers. Biomedical Engineering    Society Annual Meeting (BMES), Austin, Tex.-   El-Sherbiny I M, and Smyth H D C. (2010) Novel non-covalently    crosslinked hydrogel nano-microparticles for inhalation therapy.    Annual Meeting of American Association of Pharmaceutical Scientists,    New Orleans, La.-   El-Sherbiny I M, and Smyth H D C. (2009) Cryomilled physically    crosslinked biodegradable hydrogel microparticles as novel potential    carriers for inhalation therapy. Annual Meeting of American    Association of Pharmaceutical Scientists, Los Angles, Calif.,    AM-09-01692.-   El-Sherbiny I M, and Smyth H D C. (2009) Novel spray dried    biodegradable semi-IPN hydrogel microspheres for pulmonary drug    delivery. Annual Meeting of American Association of Pharmaceutical    Scientists, Los Angeles, Calif., (AM-09-01708).

Reference to these publications should not be taken as an admission thatthe contents of any particular document are relevant prior art. However,the skilled person is referred to each of these publications for detailsof ways in which nanoparticles and/or microparticles may be produced.

Various preliminary studies (referred to in the citations listed above)performed with regard to options for inhalation, and oral therapysupport the proposal that polymeric nanoparticles and/ornanoparticles-microparticles carrier systems will improvebioavailability, increase targeting, reduce dose frequency, avoidmacrophage clearance and confer sustained delivery of therapeutic agentsto the lung compared to free drugs. Remarkably, the preliminary data hasshown that:

(1) The loading of drugs into polymeric nanoparticles has the potentialto significantly enhance dissolution and absorption, and consequentlycan improve the bioavailability of the loaded drugs.

-   -   (2) drug-loaded nanoparticles can be incorporated into novel        crosslinked microparticles with physicochemical characteristics        (aerodynamic size, shape, moisture content, etc) appropriate for        inhalation therapy.    -   (3) the design and composition of the        nanoparticles-microparticles carriers can be modulated to allow        them to absorb moisture and expand rapidly to evade endocytosis        by macrophage cells.    -   (4) biodegradation rates of the developed carriers are        controllable.    -   (5) the polymeric nanoparticles-microparticles systems can be        used efficiently for dry powder inhalation therapy.    -   (6) crosslinked polymeric microparticles incorporating        drug-loaded nanoparticles can efficiently confer sustained        release of these drugs once deposited in the lung.

Hypothesis

The hypothesis underlying this invention is that the development ofappropriate respirable crosslinked polymeric nano-microparticle systemswill enhance bioavailability, increase deep lung targeting, reduce dosefrequency, avoid macrophage clearance and confer sustained pulmonarydelivery of Prostacyclin analogues, Nitric Oxide and PPAR β agonists forthe treatment of pulmonary arterial hypertension (PAH) compared to freedrugs. Also, that the formulation of the above into pre-designedbiodegradable and biocompatible smart pH-responsive hydrogel particleswill improve bioavailability, reduce dose frequency, increase targeting,and confer sustained oral and intravascular delivery of theseinterventions compared to free drugs.

1. Composition of Nanoparticles

Our preliminary studies showed that incorporation of drug-loadednanoparticles into respirable crosslinked microparticles can allow foradditional control of drug action and release. Also, in our preliminaryinvestigations, we found that loading of hydrophobic active ingredientsinto nanoparticles (made of ubiquitous polymers such as PLGA)considerably enhances the dissolution/absorption of these ingredients.Building on these observations, a new series of specifically designedpolymeric nanoparticles were obtained via self assembly of a new seriesof amphiphilic graft and block copolymers. The developed graft and blockcopolymers are based on natural polymers and chemically modified naturalpolymers such as, but not limited to, chitosan, chitosan derivatives,alginate, carrageenan, and cellulose derivatives.

Materials: The amphiphilic copolymers were produced via chemicalmodifications of some natural polymers such as, but not limited to,chitosan and chitosan derivatives through introducing of hydrophilicside chains (such as PEG) and/or hydrophobic moieties (mainly, stearicacid, cholanic acid, phthaloyl, and butyl acrylate). Chitosan, acationic biopolymer obtained through alkaline N-deacetylation of chitin,has been selected as a preferred base polymer for the development of thenanoparticles due to its numerous desirable characteristics includingbiodegradability, non-toxicity, biocompatibility, in addition to itsability to enhance drug absorption in lung tissues through opening theintercellular tight junctions of the lung epithelium (see Parka J H,Kwon S, Lee M, Chung H, Kim J H, Kim Y S, Park R W, Kim I S, Seo S B,Kwon I C, and Jeong S Y. (2006) Self-assembled nanoparticles based onglycol chitosan bearing hydrophobic moieties as carriers fordoxorubicin: in vivo biodistribution and anti-tumor activity.Biomaterials, 27: 119-126).

Preparation Method: The self-assembled polymeric nanoparticles wereprepared with (and without) sonication of different concentrations ofthe modified polymer solutions at different sonication powers (30-75 W)for different intervals. The effect of relative compositions plus thedifferent preparation parameters onto the physicochemicalcharacteristics (mainly particle size) of the resulting nanoparticleswere investigated extensively, to ensure desired properties have beenachieved.

Drug loading: The nanoparticles of different architectures loaded withthe bioactive moieties (treprostinil, Selexipag, PPAR β agonists or NOdonor) were prepared in the same manner used for plain nanoparticles.Then, the effect of relative compositions plus the different preparationparameters onto both drug loading capacity and loading efficiency (%) ofthe produced nanoparticles were studied in detail to determine optimalloading.

2. Nanoparticle Characterization

Polymer Characterization: The synthesized and chemically modifiedpolymers used as pre-cursors for the nanoparticles were characterizedusing different analytical tools such as elemental analysis, FT-IR,differential scanning calorimetry (DSC), and thermogravimetric analysis(TGA). Also, the crystallography patterns of powdered modified polymerswere investigated by X-ray diffraction (XRD).

Physicochemical characterization of the developed nanoparticles: Thephysicochemical properties of the developed nanoparticles such asparticle size, moisture content, and morphology were examined usingdynamic light scattering, moisture analyzer, scanning electron, andatomic force microscopy. Also, the biodegradation rates of thenanoparticles were estimated. Both plain and cargo (treprostinil,Selexipag, PPAR β agonists or NO donor)-loaded nanoparticles withoptimum physicochemical characteristics, drug loading capacity, drugrelease patterns were selected for further use in the preparation of aseries of novel respirable crosslinked polymeric nano-microparticlescarriers.

3. Microparticle Composition

Based on our preliminary studies, the developed nanoparticles must beencapsulated or formulated into microparticles for pulmonary drugdelivery due to their small aerodynamic size, which normally leads tolimited deposition in the airways and extensive exhalation from thelungs following inspiration. Also, nanoparticles-based carrier systemsmay aggregate in both dry powder and liquid forms which causes rapidclearance by macrophage cells. This aspect of the invention develops arange of novel carrier systems for controlling drug delivery (mainlypulmonary) and combines the benefits of both polymeric nanoparticles andthe respirable micron-size crosslinked hydrogel particles.

Materials: The respirable microparticles developed comprise ofsemi-interpenetrating polymeric networks (semi-IPNs) and full-IPNs.These semi- and full-IPNs are based mostly on natural polymers such as,but not limited to, chitosan and water soluble chitosan derivatives(such as carboxymethyl and PEGylated derivatives) in a combination withone or more of nontoxic, biocompatible, and biodegradable polymersincluding, but not limited to, hyaluronate, carrageenan andoligoguluronate. The semi-IPN and IPN microparticles are crosslinkedthrough ionotropic gelation, polyelectrolyte complexation and/orH-bonding. These microparticles incorporating drug (treprostinil,Selexipag, PPAR β agonists or NO donor)-loaded nanoparticles wereproduced using spray-drying technique, spray gelation, and ionotropicgelation followed by lyophilization.

4. Nano-Microparticle Characterization

The design criteria of the nano-microparticle carriers requiresgeometric and aerodynamic particle sizes for lung delivery (1-5 μmaerodynamic diameter of dry particles), rapid dynamic swelling (>20 μmwithin minutes), appropriate morphology, reasonable biodegradationrates, low moisture content, high drug (treprostinil, Selexipag, PPAR βagonists or NO donor) loading efficiency and desirable drug releaseprofiles.

The physicochemical properties of the nano-microparticles carriers, suchas particle size, moisture content, and morphology were investigatedwith the aid of dynamic light scattering (DLS), moisture balance,scanning electron microscopy (SEM), and atomic force microscopy (AFM).Particle density and dynamic swelling were determined using pycnometerand laser diffraction, respectively. The loading capacity, and releasekinetics of the incorporated treprostinil, Selexipag, PPAR β agonists orNO donor were determined. Also, the biodegradation rates of thedeveloped formulations were measured. Based on the pre-suggested designcriteria, the treprostinil-loaded nano-microparticles carriers withoptimum physicochemical characteristics, high treprostinil loadingefficiency, and desirable in vitro treprostinil release kinetics will beselected and optimized for further in vitro assessment of their deliveryperformance. Further selection and optimization of the nano-microcarriers will then determine efficacy in the treatment of PAH in an invivo (rat) model, before further development towards human trials.

Characteristics and Potential Uses

Improving the physicochemical characteristics of treprostinil viaappropriate formulation and targeting would enhance its overall targetorgan bioavailability.

Incorporation of treprostinil or other drugs suitable for treatment ofPAH in polymeric nano-micro-carriers is advantageous over other carriersbecause of stability, potential for improved permeability across thephysiological barriers, increased bioavailability and also reduction ofundesirable side effects. In addition, the polymeric nano-micro-carrierscan be designed with desirable physicochemical characteristics via theselection of appropriate candidates from a wide range of the availablenatural and synthetic polymers.

Examples (I) Preparation of Plain and Therapeutic Agent-LoadedPoly(Lactic-Co-Glycolic Acid) Nanoparticles

The plain and therapeutic agents-loaded poly(lactic-co-glycolic acid)[PLGA] nanoparticles were prepared using “single Emulsion/solventevaporation technique” through a procedure similar to that described inour previous work (see El-Sherbiny I M et al, 2010, Carbohydr Polym, 83,1345-1354). Briefly, 1 g of PLGA was dissolved in 50 ml of methylenedichloride. Then, to this PLGA solution, 5 ml of the therapeutic agent(treprostinil, selexipag, PPAR β agonists or NO donor) solution wasadded with stirring. A 2.5% w/v aqueous polyvinyl alcohol (PVA) solution(70 ml) was prepared to which, the PLGA/therapeutic agent mixture wasadded dropwise while vortexing the capping agent (PVA) solution at highsetting. The mixture was then sonicated for 2 min at 50% amplitude tocreate an oil-in-water emulsion. The sonication process was repeatedthree times until the desired size of the nanoparticles was obtained.The sonication process was performed in an ice-water bath with usingpulse function (10 s pulse on, and 10 s pulse off) in order to evade theheat built-up of the PLGA/therapeutic agent solution during thesonication. Afterwards, the emulsion was immediately poured into 100 mlof an aqueous 0.3% w/v PVA solution with rapid stirring. The resultingPLGA nano-emulsion was stirred overnight in uncovered container to allowfor methylene chloride and ethanol evaporation. The resulting PLGA NPsaqueous suspension was used directly or further used in the preparationof the nano-in-microparticles for inhalation. The prepared PLGAnanoparticles showed dense, compact, and integrated spherical shapeswith particle radius of 247±10 and 271±18 nm for the plain and thedrug-loaded PLGA nanoparticles, respectively, as determined by DLS.

(II) Preparation of Plain and Therapeutic Agent-Loaded Chitosan-BasedNanoparticles:

(1) Preparation of PEG-Drafted-CS Copolymer

The copolymer of PEG grafted onto CS was prepared (as illustrated inFIG. 1) by a modified method of that reported in our earlier study(El-Sherbiny, I. M., & Smyth, H. D. C. (2012). Controlled releasepulmonary administration of curcumin using swellable biocompatiblenano-microparticles systems. Molecular Pharmaceutics, 9(2), 269-280) anddescribed briefly as follows:

(i) Preparation of PEG-COOH: methoxy-PEG (100 g, 20 mmol),4-dimethylaminopyridine, DMAP (2.44 g, 20 mmol), triethylamine (2.02 g,20 mmol), and succinic anhydride (2.4 g, 24 mmol) were dissolved in 300ml of dry dioxane. The mixture was stirred at room temperature for 2days under a dry nitrogen atmosphere. Dioxane was then evaporated undervacuum and the residue was taken up in CCl₄, filtered and precipitatedby diethyl ether to produce PEG-COOH powder. (ii) Masking of the NH₂groups of CS: phthalic anhydride (44.8 g, 5 molequivalent to pyranoserings) was reacted with 10 g of CS in 150 ml of DMF at 130° C. underinert atmosphere for 10 h. The resulting phthaloyl CS (PhCS) was thencollected by filtration after precipitation on ice, washed extensivelywith methanol, and dried at 45° C. under vacuum to produce the yellowishbrown PhCS. (iii) Conjugation of PEG-COOH with PhCS: PEG-COOH (37.9 g)was stirred with PhCS (5.0 g, 0.4 mol equivalent to PEG-COOH) in 70 mlof DMF. Then, 1-hydroxybenzotrizole, HOBt (3.4 g, 3 mol equivalent toPEG-COOH) was added with stirring at room temperature until a clearsolution was obtained. The 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDC.HCl (4.25 g, 3 mol equivalent toPEG-COOH) was then added with stirring the mixture overnight at roomtemperature. A purified PEG-g-PhCS copolymer (5.47 g, white product) wasobtained after dialysis of reaction mixture against distilled waterfollowed by washing with ethanol. (iv) Demasking of PEG-g-PhCS:PEG-g-PhCS (4.1 g) was heated up to 100° C. with stirring under inertatmosphere in 20 ml of DMF. Then, 15 ml of hydrazine hydrate was addedand the reaction was continued for 1.5 h. The resulting PEG-g-Cs waspurified by dialysis against a (1:1) mixture of ethanol and deionizedwater then dried under vacuum at 45° C.

2. Preparation of PEG-CS-Oleic and PEG-CS-Cholanic Acid Copolymers:

Hydrophobic moieties including oleic, and cholanic acid were coupled toCS backbone of the PEG-g-CS by formation of amide linkages through theEDC-mediated reaction as follows (FIG. 2): Briefly, PEG-g-CS (1 g) wasdissolved in 0.6% (w/v) aqueous acetic acid solution (100 ml) anddiluted with 85 ml methanol. HM was then added to PEG-g-CS solution at0.4-0.5 mol/l glucosamine residue of CS followed by a drop-wise additionof 15 ml EDC methanol solution (0.07 g/l) while stirring at roomtemperature. After 20 h, the reaction mixture was added to 200 ml ofmethanol/ammonia solution (7/3, v/v) while stirring. The precipitatedmaterial was filtered; washed with distilled water, methanol, and ether;and then dried under vacuum for 20 h at room temperature. The DS, whichrepresents the number of HM groups per 100 amino groups of CS, wasevaluated using normal titration.

3. Characterization of the Modified Polymers:

The synthesized and chemically modified polymers used as pre-cursors forthe nanoparticles fabrication were characterized using severalanalytical techniques such as elemental analysis (EA), Fourier transforminfrared (FT-IR), nuclear magnetic resonance (NMR), differentialscanning calorimetry (DSC), and thermogravimetric analysis (TGA). Also,the crystallography patterns of powdered modified polymers wereinvestigated by X-ray diffraction (XRD).

4. FT-IR and Elemental Analysis Data of Some of the Developed Polymersand Copolymers

PhCS: FT-IR (v_(max), cm⁻¹) 3286, 2972, 1770, 1689, 1401, 1050, 727;(C₈H₁₃NO₅)_(0.2363)(C₆H₁₁NO₄)_(0.016)(C₁₄H₁₃NO₆)_(0.747), calculated (%)(DS=0.97) (%): C, 55.71; H, 4.86; N, 5.21. found (%), C, 60.27; H, 4.80;N 4.97. PEG-COOH: FT-IR (v_(max), cm⁻¹) 3502, 2879, 1743, 1114;(C₂₃₁H₄₆₁O₁₁₇), calculated (%): C, 54.38; H, 9.04. found (%), C, 56.3;H, 9.21. PEG-PhCS copolymer: FT-IR (v_(max), cm⁻¹) 3411, 2901, 1739,1712, 1091, 720, found EA (%), C, 56.21; H, 4.61; N, 5.22. PEG-CScopolymer: FT-IR (v_(max), cm⁻¹) 3305, 2871, 1706, 1099. found EA (%),C, 40.51; H, 4.74; N, 14.09.

5. Development of Plain and Drug-Loaded Modified CS-Based Self-AssembledNanoparticles:

The developed modified CS copolymers (PEG-g-CS, PEG-CS-Oleic, andPEG-CS-Cholanic) were used to develop a new series of self-assemblednano-carrier systems for the controlled sustained delivery oftreprostinil, selexipag, PPAR β agonists and NO. The nanoparticles wereprepared with and without sonication of different concentrations(0.05-1.5%) of the modified polymers solutions using probe sonicator.The sonication process was performed at different sonication powers(20-45 W) for different intervals (30-120 s). The effect of relativecompositions plus the different preparation parameters onto thephysicochemical characteristics (particle size, morphology, drug loadingcapacity, and moisture content) of the resulting nanoparticles wasinvestigated. The prepared self-assembled nanoparticles showed particleradius of 95±7 and 105±13 nm for the plain and the drug-loadednanoparticles, respectively, as determined by DLS.

6. Development of Plain and Drug-Loaded Modified CS-Based HydrogelNanoparticles:

The prepared CS copolymers (PEG-g-CS, PEG-CS-Oleic, and PEG-CS-Cholanic)were used to develop new series of hydrogel nano-carrier systems for thecontrolled sustained delivery of treprostinil, selexipag, PPAR βagonists and NO. This has been achieved using various types ofcrosslinkers (mainly tripolyphosphate, TPP and genipin). The preparationwas carried out in a mild aqueous media to ensure the stability of theloaded therapeutic agents (treprostinil, selexipag, PPAR β agonists andNO-donor). The prepared self-assembled nanoparticles showed particleradius of 295±19 and 311±24 nm for the plain and the drug-loadednanoparticles, respectively, as determined by DLS.

7. Development of Plain and Drug-Loaded Respirable Nano-Microparticles:

The developed PLGA, self-assembled or hydrogel nanoparticles loaded withtreprostinil, selexipag, PPAR β agonists or NO-donor were incorporatedinto respirable semi-interpenetrating polymeric networks (semi-IPNs) orfull-IPNs microparticles. These semi- and full-IPNs were based mostlyonto CS derivatives (such as N-trimethyl CS, carboxymethyl CS, andPEGylated CS) in a combination with one or more of nontoxic,biocompatible, and biodegradable polymers including, sodium alginate,hyaluronate, carrageenan and oligoguluronate. The semi-IPN and IPNmicroparticles were crosslinked through ionotropic gelation,polyelectrolyte complexation and/or H-bonding. The microparticlesincorporating drug (treprostinil, Selexipag, PPAR β agonists or NOdonor)-loaded nanoparticles were produced using spray gelation,ionotropic gelation followed by lyophilization, or spray-dryingtechnique (NANO-01A nano-spray dryer, MECC CO., LTD., Japan). Thedeveloped nano-microparticles powder showed desirable swelling extents(10-24 times of dry size) within three minutes and also showed acceptedmoisture contents as dry powders (less than 1.6%), good enzymaticdegradation rates, promising drug loading capacity (more than 90%), andconsistent sustained release of the loaded therapeutic agents.

Experiments

Nanoparticles were prepared as described above from a combination ofpolymers including chitosan of low molecular weight, PEG 400 andpolyvinyl pyrolidone (PVP), and other materials such as glucose andtetramethyl orthosilicate. Particles were either loaded with an NO donor(referred to as “NO nanoparticles”) or not (“control” or “plain”nanoparticles). Glucose was used to perform a thermal reduction of theloaded NO-donor, sodium nitrite (NaNO₂) to generate NO gas. The NOremain entrapped inside the nanoparticles powder until undergoing asustained release because of the swelling of the prepared hydrogel-basednanoparticles upon exposure to moist environment. Various nanoparticleswere prepared with different concentrations of the NO-donor, sodiumnitrite. In the following experiments, a sample referred to as“NO-Polymer 4” was used. This was prepared as follows: Sodium nitritesolution was prepared (1 g in 30 mM PBS at pH 7.5). Then, D-glucose wasadded at 40 mg D-glucose/mL of sodium nitrite solution followed byaddition of PVP (6.25 mg) with stirring. Afterwards, PEG-400 was addedat 0.5 mL PEG/10 mL of solution. Acidic chitosan solution (0.5% w/v) oflow molecular weight was then added at a ratio of 0.5 mL/10 mL of sodiumnitrite solution. In another container, acidic solution of tetramethylorthosilicate (2.5 mL/0.6 ml HCl) was prepared and sonicated in icebath, then added to the NO-donor solution (1 mL tetramethylorthosilicate/10 mL solution). The resulting mixture was then stirredand set aside for gelation, followed by freeze drying. The controlnanoparticles were prepared using the same procedures but without theNO-donor.

The nanoparticles were characterised by powder X-ray diffraction (FIG. 3a) and themogravimetric analysis (FIG. 3 b). Particles were alsoassessed for NO release over a period of 8 hours (FIG. 3 c) and NOrelease over 20 minutes from different concentrations of NOnanoparticles (0.75, 1.25, 2.5, 5, and 10 mg/ml) compared to controlplain nanoparticles (at 5 mg/ml). It can be seen that the majority of NOis released in the first 100 or so minutes, while higher concentrationsof nanoparticles release more NO.

FIG. 4 shows the effect of the NO nanoparticles used in FIG. 3 onviability (FIG. 4 a) and release of the chemokine CXCL8 (FIG. 4 b) fromendothelial cells grown from blood of healthy donors with or withoutstimulation with LPS. Nanoparticles were contacted with the cells atconcentrations of 1.5, 2.5, 5, and 10 mg/ml.

FIG. 5 shows a similar experiment to FIG. 4, using pulmonary arterysmooth muscle cells (PAVSMCs) in place of endothelial cells.

FIG. 6 shows the effect of the NO nanoparticles at differentconcentrations on the relaxation of pulmonary arteries from control mice(normoxic) and mice with pulmonary hypertension (hypoxic). Relaxation isshown as a percentage of U46619-induced contraction. It can be seen thatthe NO nanoparticles induce substantial relaxation in both mouse groups.

FIG. 7 shows the effect of control nanoparticles on viability (FIG. 7 a)and release of the chemokine CXCL8 (FIG. 7 b) from endothelial cellsgrown from blood of healthy donors with or without stimulation with LPS.Nanoparticles were contacted with the cells at concentrations of 1.5,2.5, 5, and 10 mg/ml. There is essentially no effect of thenanoparticles lacking NO.

FIG. 8 shows the effect of U46619 on pulmonary arteries from controlmice (Normoxic) and those with pulmonary hypertension (Hypoxic) (FIG. 8a), and the effect of control nanoparticles (NO-free polymernanoparticles) on relaxing pulmonary arteries from hypoxic vs normoxicmice that have been pre-contracted with U46619 (FIG. 8 b). U46619(9,11-Dideoxy-9α,11α-methanoepoxy prostaglandin F2α) is a stablesynthetic analog of the endoperoxide prostaglandin PGH2, and is avasoconstrictor that mimics the hydroosmotic effect of vasopressin.

FIG. 9 shows the effect of U46619 on contractile responses of pulmonaryarteries and aorta from control mice (normoxic) (FIG. 9 a). There is asignificant contraction of the aorta, with less contraction of thepulmonary artery. FIGS. 9 b and 9 c show respectively the effect ofcontrol nanoparticles (no NO) and NO nanoparticles on relaxing pulmonaryarteries and aorta from control mice that have been pre-contracted withU46619. There is a substantial relaxation effect from the NOnanoparticles, which is not seen with the control nanoparticles.

Thus, these experiments demonstrate that NO-releasing agents can beincorporated into nanoparticles, and that these nanoparticles willinduce relaxation of pulmonary arteries and aorta in a mouse model.

1. A pharmaceutical formulation comprising polymeric nanoparticlesencapsulated within crosslinked polymeric microparticles, wherein thepolymeric nanoparticles carry a therapeutic agent suitable for treatmentof PAH loaded within them, for use in the treatment of pulmonaryarterial hypertension (PAH).
 2. The formulation of claim 1 which is aninhalable, dry powder pharmaceutical formulation.
 3. The formulation ofclaim 1 which is an oral pharmaceutical formulation.
 4. The formulationof claim 1 which is an injectable pharmaceutical formulation.
 5. Theformulation of claim 1 wherein the nanoparticles have a mean particlediameter of from 1 to 500 nm.
 6. The formulation of claim 5 wherein themicroparticles comprise a pH responsive carrier.
 7. The formulation ofclaim 6, wherein the crosslinked microparticles have a mean particlediameter from 1 to 5 μm for inhalable formulations, and from 1 to 500μm, most preferably from 1 to 250 μm for oral formulations.
 8. Theformulation of claim 7 wherein the polymeric nanoparticles comprise achitosan or a chitosan-derivative polymer.
 9. The formulation of claim 8wherein the chitosan-derivative polymer is selected from chitosan-PEG,N-trimethyl chitosan, or a derivative of chitosan comprising a stearicacid, cholanic acid, phthaloyl, or butyl acrylate side chain.
 10. Theformulation of claim 7 wherein the polymeric nanoparticles comprisepoly(lactic-co-glycolic acid).
 11. The formulation of claim 7 whereinthe polymeric nanoparticles comprise self-assembly amphiphilic chitosanderivatives.
 12. The formulation of claim 11 wherein the polymericnanoparticles comprise self-assembly amphiphilic chitosan-PEG-Cholanicacid, or chitosan-PEG-Stearic acid or chitosan-PEG-Oleic acid.
 13. Theformulation claim 12 wherein the nanoparticles are epitheliallytargeted.
 14. The formulation of claim 13 wherein the polymericnanoparticles have a moisture content, in the dry formulation, of lessthan 2%.
 15. The formulation of claim 14 wherein the polymericnanoparticles are produced via self assembly following sonication ofamphiphilic polymer solutions.
 16. The formulation of claim 15 whereinthe crosslinked polymeric microparticles are cross-linked hydrogelpolymeric microparticles.
 17. The formulation of claim 16 wherein thepolymeric hydrogel microparticles are pH-responsive and comprisesemi-interpenetrating polymeric networks (semi-IPNs) or full-IPNs. 18.The formulation of claim 17 wherein the microparticles comprise chitosanor a water soluble chitosan derivative, such as carboxymethyl andPEGylated derivatives.
 19. The formulation of claim 18 wherein themicroparticles further comprise one or more polymers selected fromhyaluronate, carrageenan and oligoguluronate.
 20. The formulation ofclaim 19 wherein the microparticles incorporating nanoparticles areproduced using spray-drying technique, spray gelation, or ionotropicgelation followed by lyophilization.
 21. The formulation of claim 20wherein the microparticles are swellable.
 22. The formulation of claim21 wherein the microparticle is able to swell to at least 500% of theoriginal (dry formulation) size.
 23. The formulation of claim 22 whereinthe formulation is an inhalable dry powder formulation, and themicroparticle is able to swell to a larger diameter within 10 minutesfrom administration to the lungs of a patient.
 24. The formulation ofclaim 23 wherein the microparticle comprises less than 7.5% water whenin the dry formulation.
 25. The formulation of claim 24 wherein thetherapeutic agent is selected from prostacyclin synthetic analogs, PPARβ agonists and NO donors.
 26. The formulation of claim 25 wherein thetherapeutic agent is a prostacyclin synthetic analog, most preferablytreprostinil or selexipag.
 27. The formulation of claim 26 wherein theanalog is treprostinil.
 28. The use of the composition of claim 1 in themanufacture of a medicament for the treatment of PAH, preferably whereinthe microparticles comprise a pH responsive carrier.
 29. A method oftreatment of PAH, the method comprising administering a compositioncomprising polymeric nanoparticles encapsulated within crosslinkedpolymeric microparticles, wherein the polymeric nanoparticles carry atherapeutic agent suitable for treatment of PAH loaded within them to apatient in need thereof, wherein the composition is administered byinhalation, preferably wherein the microparticles comprise a pHresponsive carrier.
 30. A pharmaceutical formulation comprisingpolymeric nanoparticles, wherein the polymeric nanoparticles carry atherapeutic agent selected from prostacyclin synthetic analogs, PPAR βagonists and NO donors loaded within them and wherein either: a) thepharmaceutical formulation is injectable and the nanoparticles aretargeted to the epithelium, preferably wherein the microparticlescomprise a pH responsive carrier, b) the pharmaceutical formulation isan oral formulation, the polymeric nanoparticles are encapsulated withinsmart pH-responsive cross-linked polymeric microparticles, wherein thepH-responsive microparticles are targeted to the intestines, or c) thepolymeric nanoparticles are encapsulated within crosslinked polymericmicroparticles, wherein the nanoparticles are targeted to theepithelium.